Synthesis and cytotoxicity properties of amiodarone analogues

Article abstract of DOI:10.1016/j.ejmech.2006.12.031

Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate the structure-cytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macropha

Full text of DOI:10.1016/j.ejmech.2006.12.031

European Journal of Medicinal Chemistry 42 (2007) 861e867
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and cytotoxicity properties of amiodarone analogues
Laurent Bigler a, Carlo Spirli b, Romina Fiorotto b, Andrea Pettenazzo c,
Elena Duner d, Aldo Baritussio d, Ferenc Follath e, Huy Riem Ha e,
*
a Institute of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland
b Department of Internal Medicine, Section of Digestive Diseases Yale University, 333 Cedar Street, New Haven, CT 06519, USA
c Department of Pediatrics, University of Padova, Via Giustiniani 3, 35128 Padova, Italy
d Department of Medical and Surgical Sciences, University of Padova, Via Giustiniani 2, 35128 Padova, Italy
e Cardiovascular Therapy Research Laboratory, Clinical Research Center, University Hospital of Zurich, Ra¨mistrasse 100,
CH-8091 Zurich, Switzerland
Received 9 October 2006; received in revised form 12 December 2006; accepted 21 December 2006
Available online 14 January 2007
Abstract
Amiodarone (AMI) is a potent antiarrhythmic agent; however, its clinical use is limited due to numerous side effects. In order to investigate
the structureecytotoxicity relationship, AMI analogues were synthesized, and then, using rabbit alveolar macrophages, were tested for viability
and for the ability to interfere with the degradation of surfactant protein A (SP-A) and with the accumulation of an acidotropic dye. Our data
revealed that modification of the diethylamino-b-ethoxy group of the AMI molecule may affect viability, the ability to degrade SP-A and vac-
uolation differently. In particular, PIPAM (2d), an analogue with a piperidyl moiety, acts toward the cells in a similar manner to AMI, but is less
toxic. Thus, it would be possible to reduce the cytotoxicity of AMI by modifying its chemical structure.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Amiodarone; Amiodarone analogues; Cytotoxicity; Endocytic pathway
1. Introduction
multifactorial and may result from the accumulation of AMI
itself (and/or metabolites, including iodine), altered cellular
function, free radicals overproduction, etc. [9,10]. Further-
more, the long elimination half-life (months after oral admin-
istration) of the drug complicates clinical decisions, when
a change in treatment strategy is desired. Efforts have been
made, therefore, to modify the chemical structure of AMI in
order to obtain a better therapeutic profile [11e14].
Among side effects of AMI, pulmonary toxicity is probably
the most significant and potentially life-threatening effect as-
sociated with AMI use. In humans, this incidence was approx-
imately 6%, with a mortality rate estimated at 5e10% of
affected patients [15]. Pathologically, AMI-induced pulmo-
nary toxicity (AIPT) is characterized by the accumulation of
phospholipids (phospholipidosis) in the lungs, alveolitis,
with fibrosis developing in some patients. The principal re-
sponse of lung phospholipidosis is the ‘‘foamy’’ lung, which
is due to the appearance of large intra-alveolar cells. These
Fifty years ago, during the search for potent coronary dila-
tors, the scientists of Labaz laboratories modified the chemical
structure of khellin (Fig. 1), which is a natural product with
known coronary dilator property. Further research led to the
discovery of amiodarone (AMI) as an antianginal drug [1];
its antidysrhythmic activity was only observed later [2]. At
present, AMI is one of the most potent antiarrhythmic agents
in the treatment of ventricular and supraventricular arrhyth-
mias [3,4]. However, AMI therapy is accompanied by many
undesired effects [5e8]. The mechanism of toxicity is
* Corresponding author. Cardiovascular Therapy Research Laboratory e
Hlab10, Clinical Research Center, University Hospital Zurich, Ramistrasse
¨
100, CH-8091 Zurich, Switzerland. Tel.: þ41 44 255 37 01; fax: þ41 44
255 44 45.
E-mail address: huyriem.ha@usz.ch (H.R. Ha).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.12.031

862
L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
Table 1
Physicochemical parameters of amiodarone analogues
CH3
O
I
O
O
O
O
b
c
Solubilitya at Solubilitya at log DODS7.4
log DIAM7.4
O
Compound
pH 5.0 (mM)
pH 7.4 (mM)
N
I
O
O
C4H9
AMI
2.04
2.21
0.23
1.86
2.22
0.016
0.051
0.014
0.049
0.023
0.015
0.212
7.91
7.15
8.74
7.82
8.04
6.15
7.98
4.23
3.64
4.58
4.23
4.28
3.36
3.56
2a MeAM
2b DIPAM
2c PYRAM
2d PIPAM
H3C
Khelline
Amiodarone
Fig. 1. Chemical structure of khellin and amiodarone.
2e MOPAM 0.17
2f PPAM 1.04
cells, which appear to originate from alveolar macrophases,
contain lysosomally derived lamellar inclusions [16,17]. Alve-
olar macrophases were previously used in AMI lung toxicity
investigations [18,19].
This work aims to look for the way how to minimize the cy-
totoxicity of AMI. For this goal, a group of new AMI derivatives
with modified ethoxydiethylamine side chain was synthesized,
and their effects on alveolar macrophages [viability, release of
lactate dehydrogenase (LDH), degradation of surfactant protein
A (SP-A) and uptake of an acidotropic dye] were investigated.
The result is the mean of triplicates.
a
Solubility of amiodarone analogues at 22 ꢀC in 5 mM K2HPO4 adjusted to
pH 5.0 and pH 7.4 with dilute HCl. Methanol was used as cosolvent. See
Ref. [24].
b
Lipophilicity as measured by octadecyl silane (ODS)-RP-HPLC in 0.01 M
phosphate buffer at pH 7.4 and methanol. See Ref. [23].
c
Membrane affinity as measured by IAM-HPLC at 37 ꢀC in 0.01 M
K2HPO4 adjusted to pH 7.4 with dilute HCl. Acetonitrile was used as cosol-
vent. See Ref. [21].
a partitioning mechanism, however, the charge interaction be-
tween solutes and phospholipids is not negligible (unpublished
results).
2. Results and discussion
2.1. Chemistry
2.3. Effect of AMI analogues on alveolar macrophages
The starting product B2 (1) can be obtained with high
purity by recrystallization from ethyl carbonate. From this
compound, other target compounds may be prepared by con-
densing it with the corresponding halogenated alkylamine-
products. For this step, using an excess of halogenated
reagents and always keeping the temperature of the reaction
under 90 ꢀC are recommended.
The analogues MeAM (2a), DIPAM (2b), PYRAM (2c),
PIPAM (2d), and MOPAM (2e) have chemical structures
that are very close to that of AMI (diiodo-moiety linking to
an ethoxy-tert-amino side chain). The alkoxyamine chain of
PPAM (2f) is one eCH2 unit longer than that of MeAM (2a).
AMI main metabolite monodesethylamiodarone and other
analogues, including MeAM (2a), have been used for investi-
gating their effect on the degradation of SP-A by alveolar mac-
rophages in our previous studies [19]. We completed this study
to investigate the effect of five newly synthesized derivatives
[DIPAM (2b), PYRAM (2c), PIPAM (2d), MOPAM (2e), and
PPAM (2f)]. Using trypan blue exclusion as an index of viabil-
ity, it was observed that AMI, PYRAM (2c), and PPAM (2f) de-
creased macrophage viability at high concentrations (50 mM),
while the other analogues had no effect (Fig. 2). Considering
the release of LDH as a measure of cell damage, it appears
that PPAM (2f) and PYRAM (2c), having similar toxicity
2.2. Physicochemical properties of AMI analogues:
aqueous solubility, liposolubility and interaction with
plasma membranes using chromatography
#
AMI
#
2f PPAM
The published data support the notion that AMI is seques-
tered in plasma membranes and, hence, influence its fluidity
and also enzyme activity [20]. This prompted us to use the im-
mobilized artificial membrane (IAM) to study the affinity
(log DIAM7.4) of AMI derivatives toward plasma membranes
[21], and octadecyl silane (ODS) reversed-phase HPLC to
quantify their lipophilicity (log DODS7.4) [22,23]. Additionally,
the aqueous solubility of AMI derivatives having diverse polar
moieties was also measured at 22 ꢀC in 5 mM phosphate
buffer pH 7.4, and pH 5.0 (around the pH of lysosomes) using
the published method [24]. Our data (Table 1) suggest that: (i)
the modification of the alkoxyamine moiety of AMI, as de-
scribed herein, does not induce a great change in the physico-
chemical properties of the origin molecule; (ii) the affinity of
AMI analogues toward plasma membranes is dominated by
*
2b DIPAM
2d PIPAM
2e MOPAM
50 µM
20 µM
#
5 µM
2c PYRAM
0
20
40
60
Viability (%)
80
100
120
Fig. 2. Effect of amiodarone analogues on viability of alveolar macrophages
analyzed by trypan blue exclusion. Mean ꢁ SE; n ¼ 7e13. *Different from
amiodarone at same concentration, #different from other concentrations of
the same drug (by ANOVA).

L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
863
140
120
100
80
behaviors, were the most toxic among the compounds tested
(Fig. 3). Using the degradation of SP-A as an index of integrity
of the endocytic pathway, it appears that AMI, PYRAM (2c),
PIPAM (2d), and PPAM (2f) all inhibited the degradation of
SP-A to a similar extent, while DIPAM (2b) and MOPAM
(2e) had no effect (Fig. 4).
Furthermore, control macrophages accumulated Lysosensor
in small vesicles clustered near the nucleus, but also displayed
a diffuse staining of the cytoplasm (Fig. 5). A similar pattern
of fluorescence was seen in cells treated with B2 (1) and MO-
PAM (2e). It was previously shown that B2 (1) has no effect on
the degradation of SP-A [19]. In contrast, macrophages treated
with AMI, PYRAM (2c), PIPAM (2d), and PPAM (2f) accu-
mulated Lysosensor in a few very large vacuoles, frequently
located at the periphery of the cell, with less staining of the
cytoplasm (Fig. 5). Macrophages treated with DIPAM (2b) ac-
cumulated Lysosensor into small vesicles located near the nu-
cleus, as in control cells, displayed an intense staining of the
cytoplasm, and presented huge blisters at their periphery
(Fig. 5).
Considering the effects of analogues on viability, release
of LDH, degradation of SP-A and uptake of Lysosensor, it
appears that analogues causing the accumulation of Lyso-
sensor into large vacuoles [AMI, PYRAM (2c), PIPAM
(2d), and PPAM (2f)] were the ones that inhibit most the
degradation of SP-A, indicating that disordering of vesicular
traffic and inhibition of proteolysis may be interconnected
(compare Figs. 4 and 5). These findings reinforce the
view that high solubility at neutral pH may favor interaction
with the plasma membrane, while high solubility at pH 5.0
may elevate the accumulation in the lumen of acidic organ-
elles. It is tempting to speculate that accumulation into en-
dosomes could induce swelling by osmosis. It should be
noticed, however, that swelling might also be due to the in-
creased biogenesis, decreased budding or increased fusion of
*
*
*
*
*
2b
*
*
2e
60
MOPAM
DIPAM
PIPAM
PPAM
AMI
2d
2f
40
AMI
20
PYRAM
2c
0
0.1
1
Drug Concentration (µM)
10
100
Fig. 4. Effects of amiodarone analogues on the degradation of SP-A by alve-
olar macrophages. Suspensions of 106 cells in 1 ml ringer buffered albumin
(RBA) were incubated with different drugs for 1 h, then 1 mg of 125I-SP-A
was added, and the incubation was continued for further 2 h. At the end,
TCA-solute radioactivity was counted. Data are presented as a percentage of
degradation of control cells. Mean ꢁ SE; n ¼ 3e11. *Different from amiodar-
one at same concentration (by ANOVA).
the limiting membranes with other organelles, processes that
are under the control of diverse gene products and lipids
[25]. Thus, the mechanism by which AMI and some AMI
analogues cause swelling of late endosomes remains
unclear.
Among the analogues studied, DIPAM (2b), PYRAM (2c),
and PPAM (2f) are therefore more toxic than AMI, whereas
PIPAM (2d), a piperidyl derivative of AMI, acts on alveolar
macrophages in the same manner as AMI, although appears
to be less toxic. It is also clear that lipophilicity, or partition
into plasma membrane do not, alone or in combination, help
in predicting the toxicity of AMI analogues toward alveolar
macrophages.
AMI
3. Conclusion
In order to look for new derivatives of AMI, having less
toxicity, a series of AMI analogues were synthesized and
tested in vitro. Our data suggest that modifying the bulk of
the ethoxy-tert-amino moiety of AMI molecule, as described
in the current studies, does not change much the physicochem-
ical properties, such as lipophilicity, or affinity for membranes.
However, some of these modifications strongly influence the
effects on cells, and it appears that the AMI molecule may
be modified so as to maintain the effects on the endocytic
pathway, in particular the ability to inhibit proteolysis, but
with less in vitro toxicity.
*
2f PPAM
*
2b DIPAM
2d PIPAM
2e MOPAM
50 µM
20 µM
*
2c PYRAM
5 µM
0
10
20
30
40
50
4. Experimental
Release of LDH per hour(%)
Fig. 3. Effect of amiodarone analogues on the release of lactate dehydrogenase
(LDH) by alveolar macrophages. Values are expressed as a percentage of total
LDH released per hour. Mean ꢁ SE; n ¼ 7e10. *Different from amiodarone at
same concentration (by ANOVA).
1
All melting points are uncorrected. H, 13C, and inverse
gated 13C NMR spectra were recorded on a Bruker ARX-
300 spectrometer (operating at 300.13 MHz for 1H and at

864
L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
otherwise stated and dried using standard methods if needed.
˚
DMF was stored over 4 A molecular sieve under nitrogen.
Flash chromatography: silica gel Merck 60 (40e60 mm,
230e400 mesh). Thin layer chromatography (TLC): Merck
precoated silica gel 60-F254 plates; detection by UVat 254 nm.
4.1. Chemistry
The AMI analogues were prepared from (2-butyl-benzofu-
ran-3-yl)-(4-hydroxy-3,5-diiodophenyl)-methanone (B2, 1),
which had been synthesized previously [26], by condensing
with the corresponding halogenated compounds (Scheme 1).
The synthesis and analytical data of MOPAM (2e) were re-
ported in our previous communication [27].
4.1.1. (2-Butyl-benzofuran-3-yl)-(4-hydroxy-3,
5-diiodophenyl)-methanone (1, B2)
This compound was prepared previously (yield 60%) [26].
The data supporting its chemical structure are reported herein;
mp 146.4e146.9 ꢀC; NMR: dH [300 MHz] 0.85 (t, J 7.3 Hz,
3H, CH3), 1.27 (hex, J 7.4 Hz, 2H, CH2), 1.70 (quin, J
7.3 Hz, 2H, CH2), 2.77 (t, J 7.7 Hz, 2H, CH2), 7.25e7.37
(m, 2H, aromatics), 7.44 (d, J 7.7 Hz, H-4), 7.64 (d, J
8.0 Hz, H-7), 8.10 (s, 2H, H-20 and H60), 10.44 (br s, 1H,
OH); dC [75.47 MHz] 13.4 (CH3), 21.9 (CH2), 27.4 (CH2),
29.3 (CH2), 86.1 (C-30 and C-50), 111.1, 115.6, 120.7, 123.8,
124.7, 126.3, 134.0, 140.0 (H-C20 and H-C60), 153.0, 159.6,
164.4, 186.9 (CO); EI-MS (70 eV) m/z (%) 546 (Mꢃþ, 100),
517 (33), 504 (11), 503 (7), 420 (4), 390 (6), 376 (8), 375
(7), 373 (13), 263 (20), 250 (7), 249 (8), 221 (8), 201 (8).
4.1.2. (2-Butyl-benzofuran-3-yl)-4-[2-(dimethylamino-
ethoxy)-3,5-diiodophenyl]-methanone (2a, MeAM)
The cytotoxicity of MeAM (2a) was previously investi-
gated [19], its synthesis is reported herein as standard proce-
dure. To a mixture of B2 (1) (2 g, 3.66 mmol) and K2CO3
(3.45 g, 25 mmol) in toluene/water (2:1 v/v, total volume:
75 ml) heated to 55e60 ꢀC was added in small portions (about
0.2 g) N-dimethyl-2-chloroethylamine hydrochloride (2.6 g,
20 mmol; Aldrich, 9471 Buchs, Switzerland). After the addi-
tion, the temperature was raised to reach reflux over 30 min.
The yellow color of B2 (1) disappeared. The reaction was re-
fluxed for 1 h additionally, and the phases were separated
quickly using a separation funnel at 60 ꢀC. The toluene phase
was then washed three times with 25 ml water at this temper-
ature. The organic phase was evaporated to dryness. The res-
idue was suspended in 10 ml 5% NH3, and MeAM (2a) was
extracted three times with 15 ml toluene. The organic phase
was separated by means of centrifugation and evaporated to
dryness under reduced pressure. Then, 2 ml 10 N HCl and
15 ml of toluene were added, and the liquids were taken off
under reduced pressure at 80 ꢀC. A white solid was obtained
after three additional treatments with 10 ml toluene. The resi-
due was then crystallized from toluene and gave 1.43 g (60%)
of analytically pure MeAM$HCl: mp 189.5e189.7 ꢀC; crys-
tallized form of MeAM (2a) base may also be obtained: mp
Fig. 5. Effect of amiodarone analogues on the distribution of Lysosensor Green
DND-189 after uptake by alveolar macrophages. Cells were incubated for 16 h
with 10 mM of each test compound, exposed for 20 min to 1 mg/ml Lysosensor
and then examined by means of confocal microscopy. Arrows: blisters formed
during incubation with DIPAM (2b).
75.47 MHz for 13C) at 298 K. The samples were, unless other-
wise stated, measured in d6-DMSO, and tetramethylsilane was
used as an internal reference. The electron impact (EI)-mass
spectra (70 eV) were recorded on a Finnigan MAT95 instru-
ment and the electrospray ionization (ESI)-mass spectra on
a Bruker EsquireLC instrument. The samples, recorded using
the latter method, were dissolved in MeOH at 10ꢂ4 M, and
the spectra recorded at a 4 ml per min continuous flow in pos-
itive or negative mode. All solvents were of analytical grade
quality and were used without further purification unless

L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
865
I
I
O
O
O
(CH2) n
OH
N
R2
I
R1
I
O
O
1
2 a-f
Compound (yield in %)
n
R1
CH3
R2
Reagent
Cl-CH2-CH2-N(CH3)2
2
CH3
2a
2b
MeAM (72)
DIPAM (62)
CH(CH3)2
2
CH(CH3)2
Cl-CH2-CH2-N[CH(CH3)2]2
Cl-CH2-CH2-N
2
2c
PYRAM (70)
N
N
2d
2e
2f
2
2
Cl-CH2-CH2-N
PIPAM (64)
N
MOPAM (65)
O
Cl-CH2-CH2-N
O
CH3
Cl-CH2-CH3-CH2-N=(CH3)2
CH3
PPAM (60)
3
Scheme 1. Synthesis of amiodarone (AMI) analogues.
89e90 ꢀC. NMR: dH [300 MHz] 0.84 (t, J 7.3 Hz, 3H, CH3),
1.24 (hex, J 7.4 Hz, 2H, CH2), 1.69 (quin, J 7.4 Hz, 2H,
CH2), 2.49e2.51 (m, 3H, CH3), 2.73 (t, J 7.7 Hz, 2H, CH2),
2.82, (t, J 5.9 Hz, 2H, CH2), 3.29e3.31 (m, 3H, CH3), 4.08
(t, J 5.9 Hz, 2H, CH2), 7.27e7.39 (m, 2H, aromatics), 7.48
(d, J 7.7 Hz, H-4), 7.65 (d, J 8.1 Hz, H-7), 8.16 (s, 2H, H-20
and H60); dC [75.47 MHz] 13.3 (CH3), 21.8 (CH2), 27.4
(CH2), 29.4 (CH2), 45.4 (N-(CH3)2), 58.2 (CH2), 71.1 (CH2),
92.9 (C-30 and C-50), 111.1, 115.5, 120.7, 123.8, 124.8,
126.0, 137.9, 139.7 (H-C20 and H-C60), 153.0, 161.0, 165.3,
187.3 (CO); ESI-MS [M þ H]þ m/z (%) ¼ 618 (100).
Using the corresponding chloroalkoxylamine hydrochlo-
ride compounds purchased from Aldrich, 9471 Buchs, Swit-
zerland, DIPAM (2b), PYRAM (2c), PIPAM (2d), MOPAM
(2e), and PPAM (2f) were prepared in a similar manner
(Scheme 1).
(CH3), 18.0 (CH3), 21.9 (CH2), 27.5 (CH2), 29.3 (CH2), 30.6
(CH), 45.3 (CH2), 54.5 (CH), 68.1 (CH2), 92.1 (C-30 and C-
50), 111.2, 115.6, 120.8, 123.9, 124.9, 126.1, 138.7, 139.8
(H-C20 and H-C60), 153.1, 160.0, 165.6, 187.3 (CO); ESI-
MS [M þ H]þ m/z (%) ¼ 674 (100).
4.1.4. [4-(2-Butyl-benzofuran-3-yl)-[3,5-diiodophenyl-4-
(2-pyrrolidin-1-yl-ethoxy)phenyl]-methanone$
hydrochloride (2c, PYRAM)
Mp 93.6e95.4 ꢀC; NMR: dH [300 MHz] 0.84 (t, J 7.3 Hz,
3H, CH3), 1.26 (hex, J 7.4 Hz, 2H, CH2), 1.69 (quin, J
7.4 Hz, 2H, CH2), 1.92e2.08 (m, 4H, CH2), 2.73 (t, J
8.0 Hz, 2H, CH2), 3.22e3.32 (m, 2H, CH2), 3.72e3.74 (m,
4H, CH2), 4.34e4.37 (m, 2H, CH2), 7.26e7.39 (m, 2H, aro-
matics), 7.46 (d, J 7.7 Hz, H-4), 7.64 (d, J 8.1 Hz, H-7),
8.18 (s, 2H, H-20 and H6); dC [75.47 MHz] 13.3 (CH3), 21.8
(CH2), 22.6 (2CH2), 27.4 (CH2), 29.2 (CH2), 53.1 (CH2),
53.5 (2CH2), 68 (CH2), 92.1 (C-30 and C-50), 111.1, 115.5,
120.7, 123.8, 124.8, 126.0, 138.5, 139.7 (H-C20 and H-C60),
153.0, 160.0, 165.5, 167.3 (CO); EI-MS (70 eV) m/z
(%) ¼ 643 (Mꢃþ, <1%), 546 (4), 517 (5), 516 (17), 420 (2),
201 (3), 97 (5), 84 (6), 389 (100), 55 (3).
4.1.3. (2-Butyl-benzofuran-3-yl)-4-[2-(diisopropylamino)
ethoxy]-3,5-diiodophenyl-methanone$hydrochloride
(2b, DIPAM)
Mp 146.8e148.7 ꢀC; NMR: dH [300 MHz] 0.85 (t, J
7.3 Hz, 3H, CH3), 1.27 (hex, J 7.4 Hz, 2H, CH2), 1.40 (d,
J 6.3 Hz, 6H, CH3), 1.42 (d, J 6.3 Hz, 6H, CH3), 1.70 (quin,
J 7.4 Hz, 2H, CH2), 2.74 (t, J 7.7 Hz, 2H, CH2), 3.58e3.66
(m, 2H, CH2), 3.80e3.89 (m, 2H, CH2), 4.40 (t, J 6.9 Hz,
2H, CH2), 7.28e7.39 (m, 2H, aromatics), 7.48 (d, J 7.8 Hz,
H-4), 7.66 (d, J 7.8 Hz, H-7), 8.19 (s, 2H, H-20 and H60),
10.2 (br s, 1H, HNþ); dC [75.47 MHz] 13.4 (CH3), 16.7
4.1.5. [4-(2-Butyl-benzofuran-3-yl)-[3,5-diiodophenyl-4-
(2-piperidinoethoxy)phenyl]-methanone$hydrochloride
(2d, PIPAM)
Mp 131.3e172.0 ꢀC; NMR: dH [300 MHz] 0.845 (t, J
7.3 Hz, 3H, CH3), 1.27 (hex, J 7.4 Hz, 2H, CH2), 1.70 (quin,

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L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
J 7.4 Hz, 2H, CH2), 1.76e1.91 (m, 6H, CH2), 2.74 (t, J 7.8 Hz,
2H, CH2), 3.08e3.20 (m, 2H, CH2), 3.65e3.69 (m, 4H, CH2),
4.40e4.30 (m, 2H, CH2), 7.27e7.48 (m, 2H, aromatics), 7.47
(d, J 7.5 Hz, H-4), 7.66 (d, J 8.0 Hz, H-7), 8.18 (s, 2H, H-20
and H60); dC [75.47 MHz] 13.3 (CH3), 21.1 (CH2), 21.8
(CH2), 22.3 [(CH2)2], 27.4 (CH2), 29.3 (CH2), 52.5 [(CH2)2],
55.4 (CH2), 67.1 (CH2), 92.1 (C-30 and C-50), 111.1, 115.5,
120.7, 123.8, 124.8, 126.0, 138.5, 139.7 (H-C20 and H-C60),
153.0, 160.2, 165.6, 187.3 (CO); ESI-MS (70 eV) m/z
(%) ¼ 658 (100); EI-MS (70 eV) m/z (%) ¼ 657 (Mꢃþ,
<1%), 546 (3), 530 (6), 420 (2), 201 (2), 147 (3), 146 (2),
112 (2), 98 (100), 96 (3), 91 (2), 55 (4).
purposes. The isolated macrophages were suspended in ringer
buffered albumin (RBA; containing 145 mM NaCl, 5 mM KCl,
1.0 mM MgCl2, 2.5 mM Na2HPO4, 10 mM HEPES, 10 mM
glucose, 1.0 mg/ml bovine albumin, pH 7.4) at a concentration
of 106 cells/ml and used as suspensions after adhesion to Fal-
con plates (Becton Dickinson Labware, Meylan, France).
To study the effect of analogues on the ability of macro-
phages to degrade SP-A, alveolar macrophages (106 cells sus-
pended in 1 ml of RBA) were incubated for 1 h at 37 ꢀC in the
presence of different analogues added in 1 ml of MDSO (final
concentration 0e50 mM). Then 1 mg of 125I-SP-A was added,
and the incubation was continued for 1 h. Finally, the radioac-
tivity present in medium plus cells was measured. Degradation
of SP-A is presented as the percentage of the degradation mea-
sured in control cells after subtraction of the time 0 value
(0.3e0.5% of added radioactivity).
4.1.6. (2-Butyl-benzofuran-3-yl)-4-[3-(dimethylamino-
propoxy)-3,5-diiodophenyl]-methanone$hydrochloride
(2f, PPAM)
Mp 158.1e163.8 ꢀC; NMR: dH [300 MHz] 0.85 (t, J
7.4 Hz, 3H, CH3), 1.27 (hex, J 7.4 Hz, 2H, CH2), 1.69 (quin,
J 7.4 Hz, 2H, CH2), 2.30e2.36 (m, 2H, CH2), 2.73 (t, J
7.7 Hz, 2H, CH2), 2.82, (s, 6H, CH3), 3.34e3.42 (m, 2H,
CH2), 4.09 (t, J 5.7 Hz, 2H, CH2), 7.27e7.40 (m, 2H, aro-
matics), 7.48 (d, J 7.7 Hz, H-4), 7.65 (d, J 7.9 Hz, H-7),
8.17 (s, 2H, H-20 and H60); dC [75.47 MHz] 13.3 (CH3),
21.8 (CH2), 24.7 (CH2), 27.4 (CH2), 29.2 (CH2), 41.9 [N-
(CH3)2], 54.0 (CH2), 70.1 (CH2), 92.1 (C-30 and C-50),
111.1, 115.5, 120.7, 123.8, 124.8, 126.0, 138.2, 139.6 (H-
C20 and H-C60), 153.0, 160.3, 165.4, 187.3 (CO); ESI-MS
[M þ H]þ m/z (%) ¼ 632 (100).
The effect of analogues on trypan blue exclusion and on
release of LDH by alveolar macrophages was measured as
described previously [19].
4.3.2. Uptake of Lysosensor Green DND-189
Macrophages adhering to glass cover slides were incubated
for 16 h at 37 ꢀC in the presence of 2e10 mM test compounds
before being loaded for 20 min at 37 ꢀC with 1 mmol/l Lyso-
Sensor Green DND-189, which is an acidotropic dye that ac-
cumulates into acidic organelles as a result of protonation, and
its fluorescence intensity is proportional to acidity [29]. Con-
focal images were obtained using a Nikon Eclipse TE300 in-
verted microscope equipped with the NIH Image J 1.32J
(National Institutes of Health, Bethesda, USA).
4.2. Measurement of physicochemical properties:
aqueous solubility, lipophilicity and interaction with
plasma membranes using chromatography
4.4. Statistical analysis
The affinity of AMI analogues toward plasma membrane
(log DIAM7.4) was investigated using immobilized artificial
membrane (IAM), and acetonitrile as organic modifier [21].
The lipophilicity (log DODS7.4) of AMI analogues was mea-
sured by means of octadecyl silane (ODS) reversed-phase
HPLC, and methanol as organic modifier [22,23]. The solubil-
ity of tested compounds at 22 ꢀC in 5 mM phosphate buffer pH
7.4, and pH 5.0 (around the pH of lysosomes) was measured
by the described method [24]. The details of these experiments
are reported elsewhere (to be published). The physicochemical
parameters of tested compounds are expressed as the mean of
three independent measurements (Table 1). The precision of
estimates was <8%.
Data are presented as mean ꢁ SE. Differences between
groups were analyzed by means of ANOVA, using the Dun-
net’s test as the post hoc test for data normally distributed
and the Dunn’s test for the normally distributed. The level
of significance accepted was 5%.
Acknowledgement
The authors thank Aileen McAinsh, Ph.D., Nashville, TN,
USA, for assistance in editing the manuscript.
References
[1] R. Charlier, G. Deltour, R. Tondeur, F. Binon, Arch. Int. Pharmacodyn.
Ther. 139 (1962) 255e264.
4.3. Cytotoxicity of AMI analogues
[2] J. Van Schepdael, H. Solvay, Presse Med. 78 (1970) 1849e1850.
[3] B.A. Weinberg, W.M. Miles, L.S. Klein, J.E. Bolander, R.E. Dusman,
M.S. Stanton, J.J. Heger, C. Langefeld, D.P. Zipes, Am. Heart J. 125
(1993) 109e120.
4.3.1. Trypan blue exclusion, release of LDH, and
degradation of SP-A tests
SP-A was isolated from the surfactant obtained from a pa-
tient with alveolar proteinosis and labeled with Na125I to a spe-
cific activity of 400e600 cpm/ng, as reported previously [28].
Alveolar macrophages were obtained from rabbits. The ani-
mals were housed according to the rules set forth by the Uni-
versity of Padova for care and use of animals for scientific
[4] B.N. Singh, Am. J. Cardiol. 84 (1999) 3Re10R.
[5] R.N. Fogoros, K.P. Anderson, R.A. Winkle, C.D. Swerdlow, J.W. Mason,
Circulation 68 (1983) 88e94.
[6] J.J. Heger, E.N. Prystowsky, D.P. Zipes, Am. Heart J. 106 (1983) 931e935.
[7] B. Rigas, L.E. Rosenfeld, K.W. Barwick, R. Enriquez, J. Helzberg,
W.P. Batsford, M.E. Josephson, C.A. Riely, Ann. Intern. Med. 104
(1986) 348e351.

L. Bigler et al. / European Journal of Medicinal Chemistry 42 (2007) 861e867
867
[8] G. Dickstein, S. Amikam, E. Riss, D. Barzilai, Am. J. Med. Sci. 288
(1984) 14e17.
[9] P.J. Podrid, Ann. Intern. Med. 122 (1995) 689e700.
[20] P. Chatelain, R. Laruel, M. Gillard, Biochem. Biophys. Res. Commun.
129 (1985) 148e154.
[21] S. Ong, X. Qui, C. Pidgeon, J. Phys. Chem. 98 (1994) 10189e10199.
[22] J.F. Huber, C.A. Meijers, J.A. Hulsman, Anal. Chem. 44 (1972)
111e116.
[23] T. Braumann, J. Chromatogr. 373 (1986) 191e225.
[24] C.A. Bergstrom, K. Luthman, P. Artursson, Eur. J. Pharm. Sci. 22 (2004)
387e398.
[10] A. Vereckei,A.Blazovics,I.Gyorgy, E. Feher,M. Toth,G. Szenasi,A.Zsinka,
G. Foldiak, J. Feher, J. Cardiovasc. Electrophysiol. 4 (1993) 161e177.
[11] W. Sun, J.S. Sarma, B.N. Singh, Circulation 100 (1999) 2276e2281.
[12] B. Carlsson, B.N. Singh, M. Temciuc, S. Nilsson, Y.L. Li, C. Mellin,
J. Malm, J. Med. Chem. 45 (2002) 623e630.
[13] O.F. Sharifov, V.V. Fedorov, G.G. Beloshapko, A.V. Yushmanova,
L.V. Rosenshtraukh, J. Cardiovasc. Pharmacol. 38 (2001) 706e714.
[14] A. Manning, V. Thisse, D. Hodeige, J. Richard, J.P. Heyndrickx,
P. Chatelain, J. Cardiovasc. Pharmacol. 25 (1995) 252e261.
[15] W.J. Martin 2nd, E.C. Rosenow 3rd, Chest 93 (1988) 1242e1248.
[16] W.J. Martin 2nd, J.E. Standing, J. Pharmacol. Exp. Ther. 244 (1988)
774e779.
[25] T. Kuronita, E.L. Eskelinen, H. Fujita, P. Saftig, M. Himeno, Y. Tanaka,
J. Cell. Sci. 115 (2002) 4117e4131.
[26] H.R. Ha, B. Stieger, G. Grassi, H.R. Altorfer, F. Follath, Eur. J. Clin.
Pharmacol. 55 (2000) 807e814.
[27] H.R. Ha, L. Bigler, B. Wendt, M. Maggiorini, F. Follath, Eur. J. Pharm.
Sci. 24 (2005) 271e279.
[28] A. Baritussio, S. Marzini, M. Agostini, A. Alberti, C. Cimenti,
D. Bruttomesso, E. Manzato, D. Quaglino, A. Pettenazzo, Am. J. Physiol.
Lung Cell. Mol. Physiol. 281 (2001) L1189eL1199.
[29] K. Eto, T. Yamashita, K. Hirose, Y. Tsubamoto, E.K. Ainscow,
G.A. Rutter, S. Kimura, M. Noda, M. Iino, T. Kadowaki, Am. J. Physiol.
Endocrinol. Metab. 285 (2003) E262eE271.
[17] F.R. Costa-Jussa, B. Corrin, J.M. Jacobs, J. Pathol. 144 (1984) 73e79.
[18] C.L. Ogle, M.J. Reasor, Toxicology 62 (1990) 227e238.
[19] D. Quaglino, H.R. Ha, E. Duner, D. Bruttomesso, L. Bigler, F. Follath,
G. Realdi, A. Pettenazzo, A. Baritussio, Am. J. Physiol. Lung Cell.
Mol. Physiol. 287 (2004) L438eL447.